The invention relates to the field of the analysis of microscopic elements, and more particularly to the devices dedicated to the characterization and to the counting of microscopic elements using a light.
Here, “microscopic elements” are understood to mean any element of microscopic dimensions, and notably biological particles or cells (prokaryotes and eukaryotes).
In the field of in vitro diagnostics (notably hematological counting or flow cytometry), it is conventional to use characterization and counting devices, based on the interaction between a light and the various microscopic elements that constitute a sample, in order to obtain qualitative and quantitative information on these microscopic elements.
Among the techniques that are widely used, those known as scattering techniques, which include transmission, diffraction, reflection and refraction, and those known as photoluminescence that include fluorescence and phosphorescence may notably be mentioned. They allow, separately or in combination, information on the shape, volume, size, color, density, structure, biochemical nature or particle size distribution to notably be obtained.
The characterizations of biological elements, and principally of blood cells, obtained with the common techniques of flow cytometry, are nevertheless based on a limited number of variables and of cellular discrimination possibilities.
Each measurement principle is a relatively simple physical method but the simultaneous multiplication of these measurements induces interactions that can interfere with the latter to the point of rendering them unusable. Thus, the implementation of multiple measurements is limited by the ability to avoid these interactions.
In the hematology analyzers and flow cytometers present on the market, the elements are generally detected either electronically by the method of impedance measurement known as the Wallace Coulter method, or by an optical method (scattering, photoluminescence).
For example, a volume and a refractive index can be deduced from the results of a scattering analysis based on a single wavelength and two different observation angles. This mode of detection is for example described in the patent U.S. Pat. No. 4,735,504 from the company TECHNICON Instruments Corp. The optical system described in this patent uses the measurements of light diffracted over two different ranges of angles, which allows the volume and refractive index values of the elements to be separated by a comparison of their optical responses with those of elements calibrated in volume and refractive index. The processing of these data values is based on the theory of light scattering by a spherical particle developed by Gustav MIE and notably described at the Internet address <http://en.wikipedia.org/wiki/Mie_scattering>.
In flow cytometry, it is commonly accepted that the measurement of the diffraction over a range of angles close to the optical axis (FSC (for “forward scatter”) or on-axis diffraction) yields an indication of the volume of the microscopic elements analyzed. Furthermore, the light scattered orthogonally with respect to the optical axis (or “side scatter”, or again, scattering at 90°) is described as being representative of the internal structure of the elements. These characteristics are closely linked to the angles and wavelengths at which the measurements are effected.
It is recalled that fluorescence is a phenomenon induced when an excitable molecule returns to the ground state after excitation by light energy at one of its characteristic wavelengths. The emission of fluorescence light always occurs at a frequency lower than that of excitation. The fluorescence emission is substantially isotropic. The measurement is generally effected away from the excitation axis of the incident light and through an optical filter transmitting to the detector only the spectral band of interest.
The molecular probes used in fluorescence can be vital or supravital dyes having an intrinsic affinity for a particular type of molecules. Intercalating dyes for nucleic acids such as orange thiazole, O-auramine, Y-pyronine or others may notably be mentioned, or immunological probes composed of an antibody onto which a dye marker, generally a single or tandem fluorochrome or sometimes a nano-crystal, is attached.
This mode of marking by the implementation of immunological probes has become widespread for cytological identification and notably according to the flow cytometry techniques previously described. The patent EP 0 022 670 from the company Ortho Diagnostics Inc. describes the identification of various cells using flow cytometry by their antigenic determinants. The technique described in this patent has opened the door to the very extensive development of immunophenotyping which is now recognized as a cellular identification technique that is efficient, safe and reliable.
Multiparameter cytological analysis may nevertheless be carried out without making use of antigenic identification. Thus, in the document “Combined Blood Cell Counting and Classification with Fluorochrome Stains and Flow Instrumentation” J. of Histochem. & Cytochem Vol 24. No. 1, pp. 396-411, 1976, Howard M. Shapiro has described a general classification of blood cells by means of a multiparameter cytometer performing absorption, diffraction and fluorescence measurements, and in which the elements are brought into contact with a mixture of specific fluorescent dyes of nucleic acids and of proteins.
In parallel, the multiplication of the fluorescence measurement wavelengths in the analyzers has allowed the use of a growing number of markers and hence of antibodies, as indicated in the document by John A. Steinkamp, “A Modular Detector for Flow Cytometric Multicolor Fluorescence Measurements”, 1987, Cytometry 8: pp. 353-365, but has also very seriously increased the complexity of the devices and their implementation.
The combined use of dyes excited by a single wavelength quickly presents limitations owing to the regions of spectral overlap of their excitations and/or of their fluorescence emissions.
In order to overcome these problems of spectral overlap, use is generally made of a correction method called “compensation” which globally consists in reducing the fluorescence signal of the microscopic element analyzed by the proportional part of spectral overlap of other microscopic elements, as indicated in the document “Spectral Compensation for Flow Cytometry: Visualization Artifacts, Limitations and Caveats”, Mario Roederer 2001, Cytometry 45, pp. 194-205.
One problem induced by the compensations resides in the fact that they are calculated in average value and that, for a given family of fluorochromes, it is not possible to fix their values once and for all. Indeed, there exist wide variations in the physical properties of the fluorochromes of the same family notably depending on the origin of the supply. The same product coming from different manufacturers can thus lead to different sets of compensations. This especially poses a problem in the field of diagnostics because uncontrolled sources of supply could lead to erroneous and therefore potentially dangerous measurements. For example, the efficiency of the PE-TR transfer and the nature of the antibody employed are not sufficiently stable in order to accept a definitive software compensation as is notably explained in the article by Carleton C. Steward and Sigrid J. Stewart “Four color compensation”, Cytometry, Vol. 38, pp. 161-175, 1999.
The use of several excitation wavelengths allows a wider choice of dyes to be opened up and allows the emission spectra to be separated more easily. The various excitation wavelengths are frequently spatially separated in the measurement tank offering in this case the advantage of several independent measurements as described in the article by J. Steinkamp “Improved multilaser/multiparameter flow cytometer for analysis and sorting of cells and particles” John A. Steinkamp, Robert C. Habbersett, and Richard D. Hiebert; Review of Scientific Instruments Vol 62(11) pp. 2751-2764, November 1991. The problem with this method resides in the fact that the spatial displacement induces a temporal phase-shift in the responses and that the re-adjustment of the information must be done downstream, either by delaying the information in an analog fashion, or by re-adjusting the measurements by software means.
The multiplication of the excitation wavelengths (notably of the laser sources), together with measurements of fluorescence and of other optical parameters, leads to technological complexity and a significant difficulty in implementation as is for example described in the article entitled “Nine Color Eleven Parameter Immunophenotyping Using Three Laser Flow Cytometry”, Martin Bigos 1999, Cytometry 36, pp. 36-45.
The level of risk of error in the interpretation of the results increases with the number of parameters and the use of these instruments is reserved for very specialized technicians at the risk of obtaining results that are inappropriate and false, and therefore potentially dangerous.
One limitation of the compensations, whose level of criticality increases with the number of fluorochromes, relates to the propagation and the amplification of the noise in the fluorescence measurements and of their linear combinations which are used to correct the raw measurements.
Since no known analyzing device is entirely satisfactory, the object of the invention is therefore to improve the situation. In particular, the invention is designed to overcome the problems of compensation generally encountered in the prior art.